This invention describes a fast response pulsed Radiofrequency (RF) Electron Paramagnetic Resonance (EPR) spectroscopic technique for in-vivo detection and imaging of exogenous and endogenous free radicals, oxygen measurement and imaging and other biological and biomedical applications.
The main emphasis is the use of low dead-time resonators coupled with fast recovery gated preamplifiers and ultra fast sampler/summer/summer/processor accessory. Such a spectrometer will be practical in detecting and imaging with high resolution, free radicals possessing narrow line widths. This method avoids factors compromising the imaging speed and resolution inherent in the existing Continuous Wave (CW) EPR imaging methods, where modulation and saturation broadening and artifacts of object motion are problems.
It is also possible to perform Fourier imaging and hence to produce image contrasts based on relaxation when using special narrow line free radical probes.
The response of tumors to radiation therapy and chemotherapeutic agents depends upon the oxygen tension. Hence, for an effective cancer therapy, measurement of molecular oxygen in tumors is vital1. Also in general medicine measurement of the oxygen status of ischemic tissue in circulatory insufficiency, be it acute as in stroke or myocardial infarction, or chronic as in peripheral vascular disease associated with numerous diseases such as diabetes, hyperlipedimias, etc., becomes an important tool for assessment and treatment of diseases. Although a variety of techniques are available for measuring oxygen tension in biological systems, polarographic technique is perhaps the most widely used one in clinical applications. However, this is an invasive technique. Besides patients' discomfort, the tissue damage caused by the probe electrodes leads to uncertainty in the values measured, especially so at low oxygen concentration (&lt;10 mm Hg).
Magnetic Resonance Imaging (MRI) enjoys great success as a non invasive technique. NMR imaging, based on the perfluorinated organic compounds, has been used to study blood oxygenation of animal brains. Binding of oxygen to hemoglobin is also used in MRI of human brains to monitor oxygenation changes. However, these techniques lack sufficient sensitivity for routine applications.
Overhauser magnetic resonance imaging (OMRI), based on the enhancement of the NMR signal due to the coupling of the electron spin of an exogenously administered free radical with the water protons, is also attempted for in-vivo oxymetry. Here again the sensitivity is limited, since the organic free radicals used have low relaxivity since they don't possess the free sites for water binding as in the case of gadolinium based contrast agents. The Gd based contrast agents, however, have too short relaxation times for efficient spin polarization transfer. On the other hand, EPR oxymetry is very sensitive compared to MRI or OMRI for oxygen measurements, since it is based on the direct dipolar interaction of the paramagnetic oxygen molecule with the free radical probe.
EPR is generally performed at microwave frequencies (9 GHz). The use of microwave frequency results in substantial tissue heating, and, unfortunately, severely limits tissue penetration. Low frequency EPR has been attempted to achieve better tissue penetration. All of these studies but for the last cited one (from this lab) are done using Continuous Wave (CW) method.
Although low frequency EPR offers the potential for greater in-vivo tissue penetration, its use in continuous wave-based methods is severely limited by lack of sensitivity resulting from the physically imposed Boltzmann factor. Furthermore, sensitivity enhancement by signal averaging as done with CW methods may not be effective, since CW methods are band limited. Pulse EPR techniques, however, as presented in this application, utilize to advantage the very short electron relaxation times to enhance the signal to noise ratio in a very short time, which immediately leads to speed and sensitivity advantage in pulse EPR detection and imaging.
Further, the absence of any modulation in the FT method leads to true line widths, whereas in the CW methods finite modulation can, in the case of narrow lines, lead to artifacts and, therefore, severely limits the resolution achievable. Power saturation is another factor that extremely limits the resolution when detecting and imaging narrow line systems. Also for in vivo studies, any movement of the subject being studied poses severe problems in CW methods. Further, relaxation weighted imaging for contrast mapping is feasible mainly with the pulsed methods. Most of these advantages of pulse techniques over CW method are well established in MRI.
Application of pulse techniques to EPR has serious limitations. The very advantage of short relaxation time, which can in principle lead to virtual "real time" imaging, poses a challenge to the state of the art electronics for ultra fast excitation and data acquisition. Instrumental dead time problems become very severe, especially at low frequencies, since the ringing time constant, t=2 Q/w (where Q is the resonator quality factor and w is the carrier frequency), allows acquisition of signals only after a significant interval after excitation which can lead to loss of sensitivity.
The current invention addresses all of these problems and outlines pulsed EPR methodologies at radiofrequency region for in-vivo imaging of free radicals and oxygen measurement and imaging using suitable paramagnetic agents.
Apart from oxygen measurements, using appropriate, free radical probes, one can perform rapid imaging to map out blood vessels (for example, cardiac and cerebral angiography), study tissue characteristics and free radical metabolic intermediates in situ with or without using spin traps 21, 22 and offers also the potential to use administered paramagnetic contrast agents for imaging both normal and diseased tissues.
This invention has additional advantages as follows. Firstly, the magnetic field used is only about of 10 mT, orders of magnitude less than in MRI. Secondly, the sensitivity achievable is much higher than OMRI. Lastly, sensitivity enhancement, image resolution and imaging speed and T1 and T2 weighted imaging modalities are far superior to CW RF EPR.